Project Summary Opioid-induced respiratory depression is the main cause of death in opioid poisoning due to activation of mu opioid receptors located throughout the respiratory network in the brainstem. Currently, the primary chemical defense strategy to counter opioid-induced respiratory depression is the opioid receptor antagonist naloxone. While effective, it reverses all opioid effects simultaneously leading to uncontrolled pain and withdrawal in opioid dependent individuals. In addition, naloxone is not as effective at reversing opioid poisoning from highly potent opioids, such as fentanyl and synthetic fentanyl conjoiners. Medical countermeasures for opioid-induced respiratory depression that do not depend on the mu opioid receptor would be useful to combat respiratory depression, while leaving other opioid receptor effects intact. Based on preliminary data, we propose that countering opioid effects in the Kolliker-Fuse (KF), an important brainstem area in the control of breathing, is an effective strategy to counter opioid-induced respiratory depression. One potential mechanism to counter opioid effects in the KF is by activation of orexin-2 receptors. The goal of this supplemental project is to gather proof-of-principle data testing the hypothesis that orexin-2 receptor agonists could be developed as medical countermeasures to combat opioid poisoning. In Aim 1, we will use brain slice electrophysiology to determine if orexin agonists counter opioid inhibition of KF neurons on a cellular level. In Aim 2, we will use plethysmography in awake mice to determine if a systemically active orexin-2 agonist counters fentanyl-induced respiratory depression in vivo. If successful, the development of orexin-2 agonists as medical countermeasures for opioid poisoning could be accelerated, since these compounds are already being developed for narcolepsy.